Vol. Inactivation of Poliomyelitis Virus by "Free" Chiorinet G. M. RIDENOUR, PH.D., AND R. S. INGOLS, PH.D.* Department of Environmental Health, School of Public Health, University of Michigan, Ann Arbor, Mich. THE effect of chlorine on poliomyelitis virus has been a matter of considerable interest in recent years. Previous investigations on this subject have led to some general conclusions that, while the virus can be inactivated by exposure to chlorine, the amounts needed for this purpose are beyond the normal residual concenations carried in the disinfection of water supplies and swimming pools. Moreover, the time of contact required for disinfection was likewise beyond practical limits of application."w These and other investigations showed that in relation to most bacteria, poliomyelitis virus was decidedly more resistant to the germicidal effect of chlorine. The conclusions drawn from these studies were based upon the use of either the orthotolidine. or starch iodide method of determining residual chlorine, which at that time were the standard methods for the measurement of chlorine residuals. However, later studies have demonsated that these methods do not give a ue evaluation of the concenation of free uncombined chlorine in water or any 'other solution containing ammonia, niogenous compounds, and other organic matter. In * With technical assistance of Margaret Abendroth, Virus Laboratories of the Department of Epidemiology. The authors are also greatly indebted to Dr. Thomas Francis, Jr., Chairman of the Department of Epidemiology, School of Public Health, University of Michigan, whose coperative assistance made these studies possible. t This study was aided by a grant from the National Foundation for Infantile Paralysis, Inc. r9 the presence of such materials, chlorocompounds are formed which, though having an oxidation potential higher than orthotolidine or iodine under the conditions of the tests, also have a potential much less than that of uncombined or free chlorine. These chlorocompounds by virtue of their lesser redox potential have lesser disinfectant capacity. As a consequence, the inactivating or bactericidal effect of chlorine itself may not be uly evaluated without taking this factor into consideration, particularly in the case of the brain or cord suspensions used in studies with poliomyelitis virus. Newer knowledge on chlorine testing has now made it possible to measure more accurately the concenation of free or uncombined chlorine in the hypochlorous state in organically polluted solutions. This is done by the orthotolidine-arsenite test.4 In view of these considerations it was thought important to re-investigate () the effect of chlorine and () a chlorine bearing compound such as chlorine dioxide on the poliomyelitis virus in the light of the newer knowledge of chlorine measurements given by this test.4 The following studies show by this means the effect of " free " chlorine or its equivalent oxidation potential to orthotolidine on inactivation of poliomyelitis virus. METHOD OF INVESTIGATION The virus used was obtained from
4 AMERICAN JOURNAL OF PUBLIC HEALTH June, 94 TABLE Effect of "Free " on Inactivation of Poliomyelitis Virus from Chlorinie Gas Aim't Added P.P.M. 7 7 Legend: Experiment No. - ( minute contact) 8/7/7 7//.. 8//.. 7//4.. 8//.7. 7//.. concurrent infection in mice. Results not used in Table 4. ( minute contact) 8/7/7...7 8//....4.. concurrent infection in mice. Results not used in Table 4. r- Experiment No. A.7..9...... OT = Total by Standard Orthotolidine Reagent OTA = " Free " or hypochlorous acid 8/7/ = Example, 8 mice inoculated, a total, of 7 mice dead, symptoms of paralysis. no spec, no spec,... *....*. 8//4 8// 8// 8// 8//4 8// 8// 8// of the dead showed definite - TABLE Effect of "Free " on Inactivation of Poliomyelitis Virus from Dioxide (ClOJ Experiment No. I Experiment No. f t A Dioxide Aim't Added 7' 8 9 7'. 8 9 Legend: See Table ( minute contact) 8/7/.7 8//....7 8//..*.. 4.. 7//.7....... ( minute contact) 8/7/ 7/4/.i. 8//. * 8// r OT...4..8... 4........ 4.. p.p m. -.A- OTA.*.4.8..8..*..9.so... Results 8//4 8// 8// 8//4 8// 7//
Vol. paralyzed mice following inacerebral inoculation with the Lansing sain of poliomyelitis virus. Cords were taken from animals showing paralysis between the rd and th days after inoculation and made up into a per cent (:) suspension in physiological salt solution. The suspension was cenifuged at, r.p.m. and the cenifuged portion frozen at -7 C. until used in the test. just prior to the test the frozen suspension was thawed, diluted :, and cenifuged at- approximately, r.p.m. to eliminate as much suspended organic matter as possible. Tiation of the Material by inacerebral inoculation of mice gave a lethal end point between - and -4. The concenation of the virus suspension employed in the tests for inactivation was :. At this dilution the ph of the suspension was 7., ammonia niogen., and organic niogen, The method of eatment with POLIOMYELITIS 4 chlorine and chlorine dioxide consisted of placing ml. amounts of the (:) virus suspension in a series of ml. sterilized glass stoppered bottles. Various concenations of the chlorine or chlorine compounds in solution volumes of. to. ml. were then placed in the bottles and mixed by shaking immediately after the addition and at intervals during the period of the test. After a predetermined period of contact, or minutes, residual tests for chlorine were simultaneously made on each of the mixtures. At the same time a portion of the sample was eated with sodium thiosulfate to neualize the chlorine. This portion of the sample was used for inacerebral inoculation of mice under light anesthesia. An inoculation of. ml. was given to each mouse. A part of this same 'sample was also cultured for bacterial sterility in nuient broth. The orthotolidine arsenite method4 TABLE Effect of "Free " on Inactivation of Poliomyelitis Virus with the Use of a Mixture of and Dioxide (Proportion : on OT Basis) Mixture Total Aim't Added pp.m. (OT) 4 S 7 7 Legend: See Table p.pm. Experimnent No. I ( minute contact) 8//..... 8//..o.......7...*... ( minute contact) 8//. 8//.. *...7 OT......8.......7.. Experiment No. OTA..7 *... *.7.7.8 Results 8// 8// 8// 8// 8//
4 AMERICAN JOURNAL OF PUBLIC HEALTH June, 94 TABLE 4 Relation of Quantity of Applied, Total by OT, and "Free)" by OTA to Inactivation of the Virus Source of C Cl C + C Cla C C.+C Legend: See Table A pplie d S ( minute contact) ( minute contact) Aim't of OT for Inactivation.....8..........7 Average.8 Average Am't of OTA for Inactivation p.pm.......8 was used in testing for residual chlorine. This method gave a measurement of either the "free chlorine," combined chlorine such as chloramines, or total available chlorine. The results of inactivation were related to "free chlorine " hereinafter referred to as OTA and total chlorine referred to as OT Ėight mice were inoculated from each sample representing each concenation of applied chlorine. Daily checks were made on the mice for a period of days. Both deaths and paralysis were noted. However, no ansfers for the purpose of corroboration were made from dead mice in which paralysis was not observed. As a result, some deaths may have been preceded by paralysis occurring between inspection intervals. RESULTS The results of the studies conducted in the manner described above are shown in Tables -4 inclusive. The column labelled "results" in the tables represents the data from the animal inoculations. The first figure represents the number of animals inoculated, the second the total number of deaths, and the third those animals showing definite symptoms of paralysis. Interpretation as to effective chlorine residual was made on the basis of that amount of residual chlorine which inactivated sufficient virus so that neither deaths nor paralysis occurred. Asterisks are placed on Tables,, and at this point. It will be noted that the value taken on this basis is quite consistently only one increment of dosage from that which permitted at least one instance of definite paralysis. Table 4 shows an organization of the results in Table through with respect to the amount of chlorine applied, the OT residual and the OTA residual as related to the complete inactivation of the virus. DISCUSSION Inspection of the data in the Tables -4 shows that there is no definite relation between inactivation of virus and the amount of chlorine applied nor the orthotolidine residual measured by the sta,ndard acid orthotolidine (OT) reagent. s, measured by standard orthotolodine residuals, required for complete inactivation varied from. to. after minute
contact period. After minute contact the residuals varied from. p.pm. to. However, the relationship between inactivation of the virus and OTA residual is quite definite, particularly in the minute period of contact. Inactivation was secured with a minimum of. and a maximum of. of OTA residual in all instances regardless of the type of chlorine bearing material or the quantity of organic matter present in the suspension. The results with minute period were less consistent, owing presumably to the very short period of time and the attendant lack of homogeneity in the mixed preparations. These results are in sharp conast with previous investigations with respect to () reliability of the relationship of chlorine residual to inactivation of poliomyelitis virus, () the required concenation of residual chlorine, and () the time needed for inactivation. Whereas previous work has shown that inactivation of the virus required as senuous conditions as. of OT or starch iodide chlorine for Y hours to inactivate a :, dilution of virus suspension, these results indicate that. of OTA (" free" or uncombined) chlorine will inactivate the sain of virus used in these studies in as short a time as minutes. With a minute period of contact, from a ace to. of " free " chlorine caused inactivation. In addition, there is a critical and consistent relationship between the " free " chlorine residual and inactivation of the virus. The difference in the two instances is that previous investigations of the effect of chlorine on suspensions of poliomyelitis virus have been based upon a test which did not give the ue "free" chlorine residual, whereas the newly developed orthotolidine-arsenite test gives a much more accurate measure of this form of chlorine. This is the same " free " chlorine residual which is POLIOMYELITIS Vol. 4 thought of in connection with the " break-point" chlorination of water, but which also may occur in smaller concenations before the "breakpoint " is reached. The results from these studies show that chlorine, now in universal use for disinfection of water can be used in amounts which will result in inactivation of poliomyelitis virus and yet produce a satisfactory and palatable water. This is in conast to previous beliefs that if poliomyelitis were determined to be a water-borne disease chlorine would not be practical for inactivation. Also, the OTA test for residual chlorine now supplies a method of detecting the effective chlorine available for inactivation of the virus in eated waters. It would also seem important that methods now in operation for chlorination of community waters in general be reexamined to determine their efficiency in providing " free " chlorine. SUMMARY AND CONCLUSIONS In conast to previous studies, this investigation shows that chlorine is an effective inactivating agent for the Lansing sain of poliomyelitis virus if related. to the actual " free " or uncombined chlorine residual in solution. Previous work has been related to inactivation by the total.available residual chlorine in the medium, which may carry oxidation potentials from- just above that of orthotolidine to that of " free " chlorine, depending on the proportions of each. Such a heterogeneous mixture of chlorine and chloro-compounds would logically show, by tests for total chlorine, varying requirement with respect to amounts of chlorine residuals and contact periods for inactivation of the virus. If, however, the inactivation is related to the oxidation potential equivalent to that of free chlorine, a relatively small residual and a relatively short time of contact are
44 AMERICAN JOURNAL OF PUBLIC HEALTH June, 94 needed. On this basis the effect of chlorine on inactivation of -the virus is shown to be as follows:. A-" free " chlorine residual of approximately., by OTA, will inactivate a : dilution of the virus after a period of minute contact. With a minute contact, approximately. is required. Results of investigation by the authors, yet unpublished, on a pure culture of B. coh show the same degree of tolerance.. The amounts of " free " chlorine needed for inactivation are well within the range of practical dosages used in water eatment and swimming pool sanitation, when " break point" chlorination is employed. However, these "free " chlorine residuals may be reached even before the " break," in which case carrying the disinfection to the " break," may not be required for inactivation provided the required " free" residual is present before that point is reached.. It appears that the effectiveness of the "free " chlorine residual is independent of the type of chlorine-bearing compound as long as the equivalent oxidation potential of " free" chlorine exists. In these studies the results were approximately the same with chlorine, chlorine dioxide, or a mixture of the two, provided the material added was sufficient to give the required OTA residual. There was a slight tendency in favor of the chlorine dioxide OTA residual, but this was not outside the range of experimental error. Hence, it appears that the oxidation potential of the disinfectant system is a fundamental factor in the inactivation. This seems to be substantiated by the fact that ozone studies, while not shown in the data in this paper, show that an ozone concenation equivalent to. of free chlorine will also effectively inactivate the virus of poliomyelitis. ACKNOWLEDGMENT-The chlorine dioxide used in these studies was prepared from sodium chlorite furnished by the Matheison Alkali Works, Inc. Representatives of this company also gave valuable suggestions in the laboratory preparation of the material. REFERENCES. Kempf, J. Emerson, and Soule, Malcolm II. Proc. Soc. Exper. Biol. & Med., 44:4, 94.. Kempf, J. Emerson, Wilson, Martha F.,.Pierce, Marjorie E., and Soule, Malcolm IX. A.J.P.H., :, 94.. Trask, James D., and Paul, John R. A.J.P.H., :9, 94. 4. Gilcreas, F. Wellington, and Halliman, F. J. J. Am. Water Works A., :4, 944. Wisconsin Seamlines Department of Health The January-March Quarterly Bulletin of the Wisconsin State Board of Health outlines the reorganization of the State Department of Health. There are now five main adminisative sections, the last mentioned of which is entirely new. They are: General Adminisation, Preventable Disease, Sanitary Engineering, Maternal and Child Health, and Local Health Service. Dr. Allen Filek, formerly director of the Tuberculosis Division, will head the newly created Local Health Service Section. Following a rapidly growing end in other states, a new Cenal Statistical Service has been created in the General Adminisation Section. This division will prepare statistical material for all the other divisions and make data available for the use of public health personnel throughout the state.